Breast-feeding is believed to exhibit a series of advantages for the newborn compared to bottle-feeding. Human milk has been shown to impart several nutritional and health benefits, for example promotion of the infant's neuronal and intestinal development and fortification of the immune system 1, 2, 3. Apart from these aspects, many studies point out that human milk exhibits certain olfactory clues that foster the newborns' ability to orient themselves versus the human milk source 4, 5, 6, and that might even play an important role in the development of certain food preferences in later life 7, 8, 9, 10. Moreover, newborns are able to distinguish between human milk and cow's or artificial milk, they can separate between their own mother's Milk and that of other women, and are even able to distinguish between different types of human milk according to their individual temporal needs, that means between e.g. colostrum and mature human milk 4, 6, 11, 12. Even more interesting is that it was shown in these studies that babies prefer the odor attributes of human milk compared to those of artificial infant formula or milk products based on milk compositions of animal origin such as cow's milk etc. To date, most studies in the area of milk research dealt with the characterization of odor-active volatiles in processed animal milks, predominantly UHT or pasteurized milks 13, 14. From these, the conspecific odor compositions of the milks from for example cows, ewes, etc. cannot be deduced as odorants might be degraded during the heating and further processing steps, or might be formed as new and additional odor constituents. Several effects forming artificial non-natural flavor attributes in milk have been well-documented such as the sunlight-flavor etc. 13, 14.
On the other hand, there are some studies dealing with the identification and quantification of conspecific volatiles or odorants in fresh or raw animal milk 14. However, most of these studies are based on techniques such as gas chromatographic and mass spectrometric methodologies that do not allow any weighting of a possible sensory contribution of the respective compounds 15. Only a few studies targeted at the identification of odor-active substances in animal milk such as water buffalo and ewe's milk 16, 17, 18, 19. Among the most common volatiles were aldehydes, ketones, esters, lactones and several diverse substances such as terpenes and aromatic compounds.
Also, as for human milk, the influence of mother's diet on the milk odor profile compositions is not yet understood. For cow's milk and ewe's milk, there are a few studies characterizing the changes of milk odor profiles with regard to specific feeding regimes, but in most cases just on a sensory, not on a chemical-analytical basis 19, 20, 21.
While there is numerous evidence on the psychophysics level that human milk displays a very characteristic and individual olfactory composition, the molecular principles behind these phenomena remained relatively unclear. This was predominantly due to the fact that, on the one hand, sample size is very limited for comprehensive investigation of the odor composition and of the odor-dynamics of human milk in relation to single breastfeeding episodes. On the other hand, the commonly used techniques for odor analysis of human milk such as solvent extraction techniques or gas chromatographic analysis in coupling with mass spectrometry or flame ionization detection were very limited with regard to odor specificity.
This might be regarded as the main reason that only limited data on the chemical characterization of odor-active compounds in human milk is available to date 22. In this cited study, authors succeeded in identifying a total of five odorants which were detectable in all four analyzed breast milk samples. These compounds were the fatty-tallowy smelling (E)non-2-enal, the mushroom-like smelling oct-1-en-3-one, the caramel-like compounds 4-hydroxy-2,5-dimethyl-3(2H)-furanone and maltol, and the milky smelling 2-nonanone. All of these compounds were also detectable in formula milk. On the other hand, some additional compounds were identified in formula milk: the buttery smelling butane-2,3-dione, the cooked potato-like smelling 3-methylthio propanal (methional) and the meat-like smelling 2-methyl-3-furanthiol. In conclusion, no human milk-specific compound has been reported in this study. A second study reported a series of volatiles in human milk, with 6 esters, 13 ketones, 6 fatty acids, 2 lactones, 24 aliphatic aldehydes, 9 alcohols, 18 hydrocarbons, and 6 miscellaneous compounds 23. Authors used an isolation procedure based on simultaneous steam distillation-extraction under reduced pressure using diethyl ether as the solvent with a distillation temperature of 62 to 65° C. during two hours of extraction. Detection and identification of the volatile compounds was accomplished by means of gas chromatography combined with mass spectrometry based on comparison of mass spectral data with library MS spectra. However, in the cited study, the sensory properties and the sensory contribution or impact of the identified compounds with regard to human milk was not elucidated. Moreover, the list of detected volatiles comprises a series of substances which are, to the specialist of the field of odor research, highly unlikely to be odor-active contributors of human milk, for example toluene, n-propylbenzene, 1,2,4-trimethylbenzene, m-, p- and o-ethyltoluene, 1H-pyrrole, N-butyl-N-nitrosobutanamine, N,N-dibutylformamide etc. Identification of such volatiles is more likely to be produced either by artifacts, by drawbacks of the analytical outline (identification by comparison with mass spectra library data only) or by environmental contaminations of the human milk as it has been described in different aspects 24, 25. In conclusion, it seems highly improbable that these substances are natural human milk odor constituents that are supposed to compose the human milk aroma attributes. This is supported by the aspect that there are no common biochemical routes known that would explain the occurrence of such substances in human milk as endogenous compounds. In addition, it is not clear whether some of the given volatiles have been generated due to the thermal treatment during the analytical procedure as specified above, or have been destroyed. Therefore, it is not possible to identify potential odor contributors for human milk aroma among the identified volatiles presented in the cited study, and to rate their possible impact and contribution to human milk aroma.
So far the addition of natural odorants of human milk for improving the acceptance of nutraceutical compositions has not been described.
In order to find an optimal infant formula, wherein the amounts and composition of the odor-active substances are as dose as possible to mother's milk, the present inventor has studied the odor composition of fresh human milk by appropriate analytical tools without thermal exposition, predominantly taking into account the aspect of odor-activity. The analytical approach comprising gas chromatographic-olfactometric characterization of the odor volatiles was used that allowed the unambiguous identification of odor-active compounds even in small-scale human milk samples.
This approach offers the possibility to analyze milk from individual donors with minimal disturbance of the normal breast feeding procedure, and without the need to pool samples from a number of donors, as it was done in other studies. Also, the technique allows the analysis of separate individual milk sample portions from within one feeding session. That means the target was to develop an approach that allowed collection of a small portion of milk right at the beginning of the feeding episode, followed by one or more additional sampling events later on during the same feeding episode.
To achieve this goal, a very versatile and sensitive extraction technique for gaseous and liquid samples, the stir bar sorptive extraction (SBSE) was used 26. In SBSE, a PDMS-coated stir bar is exposed for a certain extraction time to a certain volume of sample either with or without preliminary application of derivatization techniques. After sorption of the analytes into the PDMS material, and removal of the matrix system, the analytes can be easily recovered via solvent extraction or thermo-desorption, and analyzed for example by means of high resolution-gas chromatography or liquid chromatography in combination with the respective detector systems. Compared to other sorptive sample preparation techniques such as SPME, the SBSE has several advantages such as convenient handling, high extraction capacity, very low amounts of PDMS breakdown products and many more 27. Apart from environmental investigations such as pesticide analysis and several others 27, 28, 29, first applications of SBSE have been reported for the direct analysis of e.g. benzoic acid or dicarboximide fungicides in foods and beverages 30, 31, for the elucidation of biochemical pathways, and for the analysis of odorous compounds in foods, mainly liquids 33, 34, 35. Combination with multidimensional gas chromatography using chiral chromatography systems allowed the assignment of the stereochemistry of aroma compounds in foods such as strawberries 36. In-vitro studies of biological markers, drugs, their metabolites or other artificial contaminants such as PCBs have been just recently performed on body fluids such as sperm, blood and urine 37, 38. Recently, SBSE was used in the context of a new in-vivo approach called BOSS (Buccal Odor Screening System) 39, 40. It was successfully applied for the characterization of volatile coffee and wine aroma “aftertaste” substances within the oral cavity. A variation of the system was applied to monitor the development of breath odor profiles after consumption of beer 43. The use in milk has not been described. An adopted approach for characterization of human milk odorants will be presented in the following.
Trace volatile and odorous substances that were characterized in fresh human milk with regard to their molecular composition and their sensory characteristics. The methodology was successfully applied for identification of more than forty characteristic odorants in human milk. The technique comprises a modified stir bar sorptive extraction system in combination with two-dimensional gas chromatographic separation and parallel mass spectrometric and olfactometric characterization of the analytes. The present invention shows that the technique can be used both for direct extractive sampling, but also for headspace analysis. Due to its applicability for small sample volumes, even day-to-day physiological variations in the profiles of volatile organic compounds in human milk samples, but also fluctuations within e.g. one breastfeeding episode can be monitored.
Accordingly, the object of the present invention is to provide nutraceutical compositions that are more attractive for babies and newborns.